Spray vaccines

ABSTRACT

THE INVENTION RELATES TO SPRAY VACCINES IN WHICH THE ANTIGEN MATERIAL IN A DRY FORM IS DISPERSED IN A LIQUID PROPELLANT. THE DISPERSING AGENT USED IS NOT A LIQUID AND AT THE SAME TIME NON-IONIC COMPOUND, BUT LECITHIN WHICH IS SOLID AND IONIC.

United States Patent 3,755,557 SPRAY VACCINES Jan Jacobs, Weesp,Netherlands, assignor to US. Philips Corporation, New York, N.Y. NoDrawing. Filed Aug. 27, 1971, Ser. No. 175,778 Claims priority,application Netherlands, Aug. 29, 1970, 7012832 Int. Cl. A61k 9/00 US.Cl. 424-46 ABSTRACT OF THE DISCLOSURE The invention relates to sprayvaccines in which the antigen material in a dry form is dispersed in aliquid propellant. The dispersing agent used is not a liquid and at thesame time non-ionic compound, but lecithin which is solid and ionic.

The invention relates to spray vaccines for medical and veterinary use.

British patent specifications Nos. 837,465 and 994,734 describepharmaceutical sprays in which a powdered medicament is dispersed in aliquid propellant. A medicament formulated in this manner may beintroduced into the upper respiratory passages through the pharynx inthe form of an aerosol by means of an atomizer.

According to both patents, a liquid non-ionic surfaceactive substance isused to form and maintain the dispersion.

Surprisingly we have now discovered that lecithin, which is neitherliquid nor non-ionic, may be used for dispersing dry prophylactics(antigens) in a liquid propellant.

The invention relates to spray vaccines for medical and veterinary usewhich are characterized in that dry antigens are dispersed in a liquidpropellant by means of lecithin.

The term antigen is used herein to mean both viral and bacterialantigens. It includes both killed and attenuated living viruses andbacteria and also toxoids.

Bacteria, microplasmata and viruses of various natures may be worked upinto vaccines according to the invention. We may mention variousinfluenza strains, such as A Aichi, A Japan, A Hong Kong, A England, BJohannesburg, B Massachusetts, B Netherlands, para-influenza strainssuch as types 1, 2 and 3. Adenovirus strains, for example types 3, 4 and7, meashes virus, Poliovirus, tetanus bacteria, diphteria bacteria andwhooping cough bacteria, Reovirus, infectious bronchitis virus, bovineviral diarrhea virus, horse influenza virus, Rhino-viruses,rhinopneumonitis virus, Newcastle disease virus, infectiouslaryngotracheitis virus, canine herpes virus, feline herpes virus,Miyagawanella virus, panleukopenia virus, distemper virus, rabies virus,pseudorabies virus, hogcholera virus, footand mouth disease vircus,vaccinia virus, bue tongue virus, Pasteurella multicida, cocci, such asStaphylococcus aureus, Staphylococcus albus, Streptococcus, Diplococcuspneumoniae, and further Escherichia, for example Escherichia coli,Salmonella, Corynebacteria, Actinobacillus, Hemophilus, Neisseria,Proteus, Pseudomonas, and the like.

The term vaccine as used in this specification includes both monovalentand polyvalent vaccines.

The antigens to be used in the vaccines may be obtained by the methodsknown for each particular species.

The viruses may be obtained by multiplication on incubated eggs or intissue cultures, possibly after previous attenuation in similar media orin animals. In general the bacteria are obtained from artificial culturemedia.

Virulent antigen material may be killed by known means, such asformaldehyde, fi-propiolactone, ultraviolet irradiation and heattreatment.

6 Claims 3,755,557 Patented Aug. 28, 1973 If desired, the antigenmaterial may be purified by conventional techniques.

The antigens must be Worked up in to a vaccine in the dry condition. Forthis puropse, they are subjected to a freeze-drying treatment, which maybe succeeded by a second drying treatment. The second treatment,however, is not always necessary, its use depending upon the conditionsin which the material is processed after the freezedrying treatment. Ifthe processing is such as to preclude 0 the absorption of moisture, thesecond drying treatment may be dispensed with.

The second drying treatment may be eifected by storing the antigensseveral days in a vacuum over a strongly hygroscopic agent such as, forexample, concentrated sulphuric acid, CaCO Na SO silica gel, P 0 and thelike. The treatment may be shortened by slightly raising thetemperature, for example, to the range of about 35 to 50 C.

The spray vaccines according to the invention may be obtained bydispersing dry antigen material in a propellant by means of lecithin.

Efiiciently the antigen suspension is mixed with lecithin before thefreeze-drying process. Thus, intimate mixing is simply achieved. As analternative, however, the lecithin may be added to the antigen materialafter the freeze-drying process.

The amount of lecithin required to disperse the antigen material as arule is at least 1 mg. per ml. of vaccine liquid. To ensure a higherstability, the amount of lecithin is preferably increased to -10 mg. perml. However, even greater amounts of, for example, mg. per ml. may beused. The upper limit is determined by the solubility in the liquidpropellant. For practical purposes the amount of lecithin may be chosenbetween 1 and 100 mg. per ml. and preferably between about 10 and 100mg. per ml. Obviously, in achieving a satisfactory suspension stabilitythe amount of antigen material per ml. also is significant. In any case,the amount of lecithin required may simply be determined. For thispurpose the antigen material is mixed with the liquid propellant in aratio which yields the desired concentration whilst adding, for example,10 mg. of lecithin per ml. of suspension. When a homogenous suspensionhas been obtained, the time which elapses before the suspended materialhas deposited is determined. If this time is too short, the stabilitymay be increased by adding more lecithin.

Suitable propellants are the gases which generally are used in pharmacyand which are liquid under pressure at room temperature. Examples ofsuch propellants are: halogenated hydrocarbons, such asdichloro-difluoromethane, dichlorotetrafluoroethane, trichloro-monofluoromethane, dichloromonofiuoromethane, monochlorodifluoromethane,trichlorotrifiuoro ethane, difluoro ethane, mono-chlorotrifluoro-ethane,hydrocarbons, such as butane, isobutane, propane and the like, ormixtures of these substances. Alternatively, mixtures of gases and gaseswhich are liquid at room temperature under pressure may also be used.

The invention, although of importance for vaccines in general, is ofparticular importance for vaccines which protect againt infections ofthe respiratory organs, since especially for this category the use of avaccine according to the invention enables the antigens to be conveyedto the area of attack of the infection which is to be controlled by thevaccine.

More particularly, the invention is of importance for influenzavaccines, since these aim at the most frequent infection of therespiratory organs.

The invention will be described more fully with reference to thefollowing examples.

V v 3 (l MONOVALENT VACCINE 4,000 embryonated eggs were inoculated bythe allantois route with influenza virus strain A Hongkong which wasadapted to eggs and mice according to the formula MK2E3M12E'1(MK'=monkey kidney, E=eggsallantois route, M=mouse). After beingincubated at 35 C. for 2 days the eggs were cooled to 4' C. (16 hours)and the allantoic fluid was separated. The volume obtained was 30litres.

The virus was purified by means of low-speed and highspeedcentrifugation at 1,300 and 50,000 g respectively, the latter processbeing performed in a Sharples centrifuge with a throughput of 1.5 litresper hour. The sediment of the high-speed centrifugation was re-suspendedin an isotonic phosphate buffer having a pH of 8.0. At 1,300 g someimpurities were still removed by centrifugation. For the purpose ofinactivation, 0.03% by weight and subsequently 0.02% by weight offl-propiolactone was added. The volume, 5 litres, was dialysed againstwater for three days with the use of a 10-fold volume per litre. Thedialysis liquid was renewed once.

To the dialysate soya lecithin in an amount of 4 mg. per 100 CCA ofvirus (CCA=chicken cell agglutination) was added. The assembly wasfreeze-dried in a Leyboldt type 904 freeze-drier. The obtained drymaterial was subjected to a second drying treatment over P in a vacuum,after which the dry substance was suspended in 40% by volume ofdichlorodifiuoromethane and 60% by volume of trichloromonofiuoromethane.The resulting product contained 1,500 CCA virus per ml.

(2) BIVALENT VACCINE 4,000 embryonated eggs were inoculated by theallantois route with influenda virus of the strain A Ainchi which hadbeen adapted to eggs according to the formula E After being incubated at35 C. for 2 days, the eggs were cooled at 4 C. (16 hours) and theallantoic fluid was separated. The volume obtained was 30 litres. Thevirus was purified by means of low-speed and high-speed centrifugationat 1,300 and 50,000 g, respectively, the latter centrifugation beingcarried out in a Sharples centrifuge having a throughput of 1.5 litresper hour. The sediment of the high-speed centrifugation was re-suspendedin an isotonic phosphate buffer having a pH of 8.0 and containing 0.01 Mof citrate. At 1,300 g some impurities were still removed bycentrifugation. For the purpose of inactivation first 0.03% by weightand then 0.02% by weight of 8- propiolactone was added. The volume, 5litres, was dialysed against water for three days with the use of a foldvolume of water. The dialysis liquid was renewed once.

3,000 embryonated eggs were inoculated by the allantois route withinfluenza virus strain B Massachusetts which had been adapted to eggs bythe Formula E After being incubated at 33 C. for 3 days the eggs werecooled and the allantoic fluid was separated. The volume was 22 litres.The virus was purified by means of lowspeed and high-speedcentrifugation at 1,300 and 50,000 g respectively, the lattercentrifugation being carried out in a Sharples centrifuge having athroughput of 1.5 litres per hour. The sediment of the high-speedcentrifugation was resuspended in an isotonic phosphate buffer having apH of 8.0. At 1,300 g some impurities were still removed bycentrifugation. For the purpose of inactivation first 0.03% by weightand then 0.02% by weight of B-propiolactone was added. The volume, 3.5litres, was dialysed against water for three days with the use of a10-fold volume of water. The dialysis liquid was renewed once.

After the dialysis the two pools were mixed. The ratio of the amounts ofvirus of strain A and strain B was 3:2.

Per 100 CCA virus 1 mg. of lecithin was added. The assembly wasfreeze-dried in a Leyboldt type G04 freezedrier, after which the drysubstance was suspended in 40% by volume of dichlorodifluoromethane andby volume of trichloromonofluoromethane. The resulting product contained1,500 CCA of A virus and 1,000 CCA of B virus per ml.

What is claimed is:

1. A spray vaccine for medical and veterinary use consisting essentiallyof a dry antigen dispersed as a stable suspension in a pressurizedliquid form of a propellant gas with lecithin as suspension stabilizer.

2. The spray vaccine of claim 1 wherein the antigen is useful againstinfections of the respiratory organs.

3. The spray vaccine of claim 2 wherein the antigen is an influenzaantigen.

4. The spray vaccine of claim 2 wherein at least 1 mg. of lecithin perml. is present.

5. The spray vaccine of claim 4 wherein from 1 to mg. of lecithin perml. is present.

6. The spray vaccine of claim 5 wherein from 10 to 100 mg. of lecithinper ml. is present.

References Cited UNITED STATES PATENTS 3,551,558 12/1970 Takebe et a1.42446 2,959,325 11/1960 Beard 424-46 3,378,443 4/1968 Cooper et a1 424463,594,471 7/1971 Hertzberger et al 424-89 3,038,816 6/ 1962 Drell et al.252-305 SHEP K. ROSE, Primary Examiner U.S. Cl. X.R.

